Development and validation of a SYBR Green I-based real-time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials

Transfusion
Mikiko SoejimaYoshiro Koda

Abstract

Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method. A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HP(del) zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates. Allelic discrimination of HP(del) yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method. The detection rate of the HP(del) allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-t...Continue Reading

References

May 10, 1984·Nature·N MaedaO Smithies
Apr 16, 1998·American Journal of Human Genetics·Y KodaH Kimura
Jul 28, 2006·Anesthesiology·Noriko ShimodeAkira Kokubunji
Feb 27, 2007·Transfusion·Ted Eastlund
Mar 17, 2007·Annals of Clinical Biochemistry·Amir H Abdul Latiff, Michael A Kerr
May 4, 2007·International Journal of Laboratory Hematology·Kymberley Carter, Mark Worwood
May 26, 2007·Transfusion·Aaron A R TobianPaul M Ness
Sep 21, 2007·Transfusion·Mikiko SoejimaHaruo Takeshita
May 30, 2008·Clinical Chemistry·Mikiko Soejima, Yoshiro Koda
Sep 13, 2008·Clinical Chemistry·Mikiko Soejima, Yoshiro Koda

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Citations

Apr 19, 2011·The Journal of Molecular Diagnostics : JMD·Mikiko SoejimaYoshiro Koda
Aug 18, 2009·Clinica Chimica Acta; International Journal of Clinical Chemistry·Hitomi NakamuraYoshiro Koda

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